Screening method

ABSTRACT

The invention discloses a screening method for the identification of new compounds for use in the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2014/067476, filed Aug. 15, 2014, which claims the benefit ofpriority under 35 USC 119(a) to European patent application numberEP13180825.5, filed Aug. 19, 2013, the disclosure of each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 18, 2016, isnamed P31257-US and is 79,177 bytes in size.

FIELD OF THE INVENTION

The present invention relates to a screening method for theidentification of compounds useful in the treatment of cancer andcompounds identified by the method of the invention.

BACKGROUND

FoxM1 is a transcription factor of the Forkhead family. It is also knownin the literature as Trident (in mouse), HFH-11 (in human), WIN or INS-1(in rat), MPP-2 (partial human cDNA) or FKHL-16. The Forkhead familycomprises a large number of transcription factors defined by a conservedDNA binding domain called Forkhead or winged-helix domain. The FoxM1gene was cloned by screening cDNA libraries with degenerate primers forhomologues with a conserved Forkhead DNAbinding domain (W. Korver, J.Roose, H. Clevers, Nucleic Acids Res. 25 (1997) 1715-1719). The FoxM1gene was revealed to encode a Forkhead transcription factor familymember that exhibits 45% identity in the DNA-binding domain with five ofits closest related Forkhead members, namely FoxA3 (HNF-3γ, FoxC1(fkh-1), FoxF2 (FREAC-2), FoxK1 (ILF) and FoxN2 (HTLF). The FoxM1C-terminal region was found to have homology (76% identity) with a humanpartial cDNA encoding an open reading-frame of 221 amino acids, termedMPP-2. MPP-2 stands for MPM-2-reactive phosphoprotein-2 and wasidentified after screening a lymphoblast-derived cDNA library with theMPM-2 monoclonal antibody, which binds specifically to epitopes onmitotic proteins that are phosphorylated in a phosphoserine-prolinedependent manner. FoxM1 binds DNA in vitro through the consensus siteTAAACA. This motif shares the core sequence recognized by other membersof the forkhead family. In particular, repeats of these motifs, inalternating orientation, were often characterized within the selectedbinding sequences for FoxM1.

The human FoxM1 gene is a 10-exon structure spanning approximately 25 kbon the 12p13-3 chromosomal band (telomeric position) (W. Korver, J.Roose, H. Clevers, Nucleic Acids Res. 25 (1997) 1715-1719). Two exons,named exons Va and VIIa, also referred to as exon A1 (or rat exon 6) andA2 respectively, are alternatively spliced (H. Ye, T. F. Kelly, U.Samadani, L. Lim, S. Rubio, D. G. Overdier, K. A. Roebuck, R. H. Costa,Mol. Cell Biol. 17 (1997) 1626-1641). Exon Va encodes a 15 amino-acidinsertion within the C-terminal part of the DNA binding-domain, and isnot seen in any of the other Forkhead transcription factor familymembers. Exon VIIa represents a 38 amino-acid insertion within theC-terminus of the protein. Differential splicing of exons Va and VIIa inhuman FoxM1, gives rise to three classes of transcripts, class Acontaining both alternative exons, class B containing none of thealternative exons, and class C in which exon Va only is retained (H. Ye,T. F. Kelly, U. Samadani, L. Lim, S. Rubio, D. G. Overdier, K. A.Roebuck, R. H. Costa, Mol. Cell Biol. 17 (1997) 1626-1641). Both FoxM1Band FoxM1C are transcriptionally active, whereas FoxM1A istranscriptionally inactive, due to the insertion of exon VIIa in theC-terminal transactivation domain. This disruption of thetransactivation domain in FoxM1A not only leads to transcriptionalinactivation, it might also cause this variant to act as adominant-negative variant as it has retained normal DNA binding activityin the absence of a functional transactivation domain (H. Ye, T. F.Kelly, U. Samadani, L. Lim, S. Rubio, D. G. Overdier, K. A. Roebuck, R.H. Costa, Mol. Cell Biol. 17 (1997) 1626-1641).

FoxM1 is overexpressed in a broad range of tumor types, including thoseof neural, gastrointestinal, and reproductive origin (see Bektas et al.,supra; Nakamura et al., 2004, Oncogene 23: 2385-400; Pilarsky et al.,2004, Neoplasia. Q: 744-50; Liu et al., 2006, Cancer Res 66: 3593-602).This expression pattern of FoxM1 is attributed to the ability of FoxM1to transactivate genes required for cell cycle progression (Wang et al.,2002, Proc Nat. Acad Sci USA 99:16881-6). Increased nuclear staining ofFoxM1B found in human basal cell carcinomas suggests that FoxM1 isrequired for cellular proliferation in human cancers (Teh et al., 2002,Cancer Res. 62: 4773-80). The detailed role of FoxM1 in establishing orfacilitating tumor progression and disease management has not been fullyelucidated, however.

EP 2 298 896 discloses siRNA molecules inhibiting expression of FoxM1Bprotein and the use of the siRNA molecules for inhibiting tumor growth.

WO 2011/127297 discloses a composition comprising a FoxM1 inhibitor andHerceptin for the treatment of breast cancer. The inhibitor is forexample a FoxM1 specific siRNA or a thiazole antibiotic such asthiostrepton.

The problem to be solved by the present invention was to provide newcompounds for the treatment of cancer.

SUMMARY

In a first aspect the present invention provides compounds inducingalternative splicing of the FoxM1 gene (splicing modifiers) for use inthe prophylaxis or treatment of cancer, wherein the compound induces atranscriptionally inactive FoxM1 variant.

In a particular embodiment, the transcriptionally inactive FoxM1 variantis FoxM1A.

In a particular embodiment, the FoxM1 gene is the human FoxM1 gene.

In a particular embodiment, the cancer is selected from the groupconsisting of cancer of the liver, prostate, brain, breast, lung, colon,pancreas, skin, cervix, ovary, mouth, blood and nervous system.

In a particular embodiment, the FoxM1 splicing modifier for use in theprophylaxis or treatment of cancer is a compound of formula I:

wherein R¹ is selected from aryl, heteroaryl, heterocycloalkyl, whichall three substituents are optionally substituted by C₁₋₇ alkyl, C₁₋₇alkoxy, C₁₋₇ haloalkoxy, C₁₋₇haloalkyl, halogen, hydroxyl, cyano, NO₂;

R² is C₁₋₇ alkoxy optionally substituted by heterocycloalkyl, NR′R″, orheterocycloalkyl optionally substituted by hydroxy, NR′R″—C₁₋₇ alkyl,hydroxy-C₁₋₇ alkyl, C₃₋₈ cyclopropyl, heterocycloalkyl, C₁₋₇ alkoxy-C₁₋₇alkyl, hydroxy-C₁₋₇ alkoxy-C₁₋₇ alkyl, halogen or azaspirocycloalkyl,azabicyloalkyl, C₂₋₇ alkynyl optionally substituted by NR′R″, orheteroaryl optionally substituted by C₁₋₇ alkyl,

R³ is halogen, C₁₋₇ alkyl,

R′ and R″ are independently selected from hydrogen, C₁₋₇ alkyl,hydroxy-C₁₋₇ alkyl.

In a particular embodiment, the FoxM1 splicing modifier for use in theprophylaxis or treatment of cancer is a compound of formula (I), whereinR¹ is aryl or heteroaryl both substituents optionally substituted byC₁₋₇ alkyl, C₁₋₇ haloalkyl, halogen, C₁₋₇ alkoxy, NR′R″, R² isheteroaryl or heterocycloalkyl both substituents optionally substitutedby C₁₋₇ alkyl, hydroxy-C₁₋₇ alkyl, halo-C₁₋₇ alkyl, R³ is C₁₋₇ alkyl.

In a particular embodiment the invention relates to compounds of formula(I), wherein:

R¹ is phenyl, imidazo[1,2-a]pyrazinyl, pyrazolo[1,5-a]pyrazinyl,imidazo[1,2-a]pyridinyl, 1,3-benzoxazolyl, indazolyl.

In a particular embodiment, the FoxM1 splicing modifier for use in theprophylaxis or treatment of cancer is a compound of formula (I), whereinR² is piperidinyl, morpholinyl, piperazinyl, pyridinyl,1,2,3,6-tetrahydropyridinyl, pyrrolidinyl.

The present invention further provides the use of a compound of thepresent invention for the preparation of a medicament for theprophylaxis or treatment or of cancer.

In a further aspect the present invention provides a pharmaceuticalformulation comprising a compound of the present invention.

In a further aspect the present invention provides a method for theprophylaxis or treatment of cancer comprising administering an effectiveamount of a compound of the present invention to a subject in needthereof.

In a further aspect the present invention provides a method of screeningfor compounds for the prophylaxis or treatment of cancer comprising:

-   -   a) contacting proliferating cells expressing the FoxM1 gene with        a test compound,    -   b) measuring the FoxM1 variant FoxM1A in the cells of step a),        wherein an increased level of the FoxM1A variant compared to a        control is indicative for a compound for the prophylaxis or        treatment of cancer.

In a further aspect the present invention provides a method of screeningfor compounds for the prophylaxis or treatment of cancer comprising:

-   -   a) contacting proliferating cells expressing the FoxM1 gene with        a test compound,    -   b) measuring the FoxM1 variant FoxM1B and/or variant FoxM1C in        the cells of step a), wherein a decreased level of the variant        FoxM1B and/or variant FoxM1C compared to a control is indicative        for a compound for the prophylaxis or treatment of cancer.

In a particular embodiment of the method of the present invention thecells are fibroblasts.

In a particular embodiment of the method of the present invention theFoxM1 variants are measured on RNA level.

In a particular embodiment of the method of the present invention theFoxM1 variants are measured on protein level.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B and 1C. Induction of alternative splicing of FoxM1 towardsFoxM1A in fibroblasts. Human fibroblasts were incubated with cpds. 1-4at different doses for 24 hours, and changes in FoxM1A RNA (containingexon A2) and FoxM1B/C (lacking exon A2) mRNA expression were assessed byRT-qPCR. Dose response curves were fitted to a Hill binding equation toestimate the EC50. FIG. 1A., upregulation of FoxM1A mRNA; FIG. 1B.,downregulation of FoxM1B/C mRNA; FIG. 1C., Correlation of EC50 valuesfor FoxM1A upregulation and FoxM1B/C downregulation. Data representmeans±SEM of 4 independent observations.

FIGS. 2A, 2B and 2C. Induction of proliferation arrest and cell death bycpds. Alternating FoxM1 splicing towards FoxM1A in fibroblasts. Humanfibroblasts were incubated with cpds. 1-4 at different doses for 5 days(120 hours), and changes in cellular impedance (Cell Index) wereassessed online. Data are expressed as delta Cell Index afternormalization for the starting value of each well. Data at 72, 96 and120 hour time points (see arrows) were averaged for quantification. FIG.2A., Micronuclei induction (MNT) in fibroblasts with increasing doses ofcpd. 1-4; FIG. 2B., Dose-dependent induction of cytotoxicity by cpds.1-4. expressed as % of untreated control after 24 hours. A cut-off at75% (ED75%, dashed line) defined the concentration at which a meaningfulimpact on proliferation and cell survival was observed. FIG. 2C.,Dose-dependent reduction of Cell Index by cpds. 1-4. expressed as % ofuntreated control. A cut-off at 75% (EC75%, dashed line) defined theconcentration at which a meaningful impact on proliferation and cellsurvival was observed. Data represent means±SEM of 4 independentobservations.

FIGS. 3A, 3B and 3C. Induction of alternative splicing towards FoxM1Acorrelates with impact on micronucleus induction and cell death infibroblasts. EC50 values for downregulation of FoxM1 ΔA2 variants and2%, EC75% and ED75% for MNT, cytotoxicity and Cell Index obtained inFIG. 1A were correlated. FIG. 3A., Correlation of micronucleus induction(2%, M) with EC50 (FoxM1ΔA2); FIG. 3B., Correlation of cytotoxicity(ED75%) with EC50 (FoxM1ΔA2). FIG. 3C., Correlation of Cell Index(EC75%) with EC50 (FoxM1 ΔA2). Data represent means±SEM of 4 independentobservations.

FIGS. 4A, 4B and 4C. Induction of alternative splicing towards FoxM1B/Ccorrelates with impact on micronucleus induction and cell death infibroblasts. EC50 values for upregulation of FoxM1 FL variant and 2%,EC75% and ED75% for MNT, cytotoxicity and Cell Index, respectively,obtained in FIG. 1B were correlated. FIG. 4A., Correlation ofmicronucleus induction (2%, M) with EC50 (FoxM1 FL); FIG. 4B.,Correlation of cytotoxicity (ED75%) with EC50 (FoxM1 FL). FIG. 4C.,Correlation of Cell Index (EC75%) with EC50 (FoxM1 FL). Data representmeans±SEM of 4 independent observations.

FIGS. 5A, 5B and 5C. Upregulation of FoxM1A protein correlates withcytotoxicity in myoblasts. Human myoblasts were treated with cpd. 3(0.1, 1 and 10 μM) for 5 days under proliferating conditions (in thepresence of serum), and total protein extracts analyzed by SDS PAGE andWestern Blot for FoxM1A and actin protein levels. FIG. 5A., FoxM1A andactin Western blots from samples treated without (control) or with cpd.3 (0.1, 1 and 10 μM); FIG. 5B., Protein levels of actin (surrogate forcell number); FIG. 5C., FoxM1A/actin ratio. Data represent means±SEM of3 independent observations. Statistical comparison was performed byone-way ANOVA followed by Dunnet's post-hoc test. *, p<0.05, **, p<0.01,***, p<0.001.

FIGS. 6A and 6B. No change in FoxM1A protein and no cytotoxicity innon-proliferating myoblasts. Human myoblasts were treated with cpd. 3(0.1, 1 and 10 μM) for 5 days under differentiating conditions (in theabsence of serum), and total protein extracts analyzed by SDS PAGE andWestern Blot for FoxM1A and actin protein levels. FIG. 6A., FoxM1A andactin Western blots from samples treated without (control) or with cpd.3 (0.1, 1 and 10 μM). Note that FoxM1A protein was undetectable; FIG.6B., Protein levels of actin. Data represent means±SEM of 3 independentobservations. Statistical comparison was performed by one-way ANOVAfollowed by Dunnet's post-hoc test.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The term “FoxM1 polypeptide” is used herein to refer to native FoxM1polypeptide from any animal, e.g. mammalian, species, including humans,and FoxM1 variants. The amino acid sequence of human FoxM1A polypeptideis given in Seq. Id. No. 1, the amino acid sequence of human FoxM1B isgiven in Seq. Id. No. 2 and the amino acid sequence of FoxM1Cpolypeptide is given in Seq. Id. No. 3.

The nucleotide sequences of the three FoxM1 variants are set forth inSeq. Id. No. 4 (FoxM1A), Seq. Id. No. 5 (FoxM1B) and Seq. Id. No. 6(FoxM1C).

The term “compound modifying splicing of the FoxM1 gene” is used hereinto refer to compounds which lead to the production of transcriptionallyinactive forms of the FoxM1 polypeptide, in particular to the productionof FoxM1A variant, by modifying the FoxM1 splicing such thattranscriptionally inactive forms are generated, in particular FoxM1A,and by suppressing the production of transcriptionally active FoxM1variants, in particular FoxM1B and FoxM1C.

The term “compound” is used herein in the context of a “test compound”or a “drug candidate compound” described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically or from natural sources. Thecompounds include inorganic or organic compounds such aspolynucleotides, lipids or hormone analogs that are characterized byrelatively low molecular weights. Other biopolymeric organic testcompounds include peptides comprising from about 2 to about 40 aminoacids and larger polypeptides comprising from about 40 to about 500amino acids, such as antibodies or antibody conjugates.

Methods for detection and/or measurement of polypeptides in biologicalmaterial are well known in the art and include, but are not limited to,Western-blotting, Flow cytometry, ELISAs or RIAs, or various proteomicstechniques. An example for a method to measure a polypeptide is anELISA. This type of protein quantitation is based on an antibody capableof capturing a specific antigen, and a second antibody capable ofdetecting the captured antigen. The assays mentioned hereinbefore aredescribed in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual,(1988), Cold Spring Harbor Laboratory Press.

Methods for detection and/or measurement of RNA in biological materialare well known in the art and include, but are not limited to,Northern-blotting, RNA protection assay, RT PCR. Suitable methods aredescribed in Molecular Cloning: A Laboratory Manual (Fourth Edition) ByMichael R. Green, Joseph Sambrook, Peter MacCallum © 2012, 2,028 pp,ISBN 978-1-936113-42-2.

The term “compound(s) of this invention” and “compound(s) of the presentinvention” refers to compound(s) modifying splicing of the FoxM1 gene,in particular to compounds of formula (I), and stereoisomers, tautomers,solvates, and salts (e.g., pharmaceutically acceptable salts) thereof.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-di ethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperizine, piperidine,N-ethylpiperidine, and polyamine resins.

The term “alkoxy” denotes a group of the formula —O—R′, wherein R′ is analkyl group. Examples of alkoxy moieties include methoxy, ethoxy,isopropoxy, and tert-butoxy.

The term “alkoxyalkyl” denotes an alkyl group wherein at least one ofthe hydrogen atoms of the alkyl group has been replaced by an alkoxygroup. Exemplary alkoxyalkyl groups include 2-methoxyethyl,3-methoxypropyl, 1-methyl-2-methoxyethyl,1-(2-methoxyethyl)-3-methoxypropyl, and1-(2-methoxyethyl)-3-methoxypropyl.

The term “alkyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 1 to 12 carbon atoms. In particular embodiments,alkyl has 1 to 7 carbon atoms, and in more particular embodiments 1 to 4carbon atoms. Examples of alkyl include methyl, ethyl, propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, or tert-butyl.

The term “alkynyl” denotes a monovalent linear or branched saturatedhydrocarbon group of 2 to 7 carbon atoms comprising one, two or threetriple bonds. In particular embodiments alkynyl has from 2 to 4 carbonatoms comprising one or two triple bonds. Examples of alkynyl includeethynyl, propynyl, prop-2-ynyl, isopropynyl, n-butynyl, and iso-butynyl.

The term “aryl” denotes a monovalent aromatic carbocyclic mono- orbicyclic ring system comprising 6 to 10 carbon ring atoms. Examples ofaryl moieties include phenyl and naphthyl.

The term “cycloalkyl” denotes a monovalent saturated monocyclic orbicyclic hydrocarbon group of 3 to 10 ring carbon atoms. In particularembodiments cycloalkyl denotes a monovalent saturated monocyclichydrocarbon group of 3 to 8 ring carbon atoms. Bicyclic means consistingof two saturated carbocycles having one or more carbon atoms in common.Particular cycloalkyl groups are monocyclic. Examples for monocycliccycloalkyl are cyclopropyl, cyclobutanyl, cyclopentyl, cyclohexyl orcycloheptyl. Examples for bicyclic cycloalkyl arebicyclo[2.2.1]heptanyl, or bicyclo[2.2.2]octanyl.

The term “haloalkoxy” denotes an alkoxy group wherein at least one ofthe hydrogen atoms of the alkoxy group has been replaced by same ordifferent halogen atoms, particularly fluoro atoms. Examples ofhaloalkoxyl include monofluoro-, difluoro- or trifluoro-methoxy, -ethoxyor -propoxy, for example 3,3,3-trifluoropropoxy, 2-fluoroethoxy,2,2,2-trifluoroethoxy, fluoromethoxy, or trifluor-methoxy. The term“perhaloalkoxy” denotes an alkoxy group where all hydrogen atoms of thealkoxy group have been replaced by the same or different halogen atoms.

The term “haloalkyl” denotes an alkyl group wherein at least one of thehydrogen atoms of the alkyl group has been replaced by same or differenthalogen atoms, particularly fluoro atoms. Examples of haloalkyl includemonofluoro-, difluoro- or trifluoro-methyl, -ethyl or -propyl, forexample 3,3,3-trifluoropropyl, 2-fluoroethyl, 2,2,2-trifluoroethyl,fluoromethyl, or trifluoromethyl. The term “perhaloalkyl” denotes analkyl group where all hydrogen atoms of the alkyl group have beenreplaced by the same or different halogen atoms.

The term “heteroaryl” denotes a monovalent aromatic heterocyclic mono-or bicyclic ring system of 5 to 12 ring atoms, comprising 1, 2, 3 or 4heteroatoms selected from N, O and S, the remaining ring atoms beingcarbon. Examples of heteroaryl moieties includepyrazolo[1,5-a]pyrazinyl, pyrrolyl, furanyl, thienyl, imidazolyl,oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl,pyridinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, triazinyl,azepinyl, diazepinyl, isoxazolyl, benzofuranyl, isothiazolyl,benzothienyl, indolyl, isoindolyl, isobenzofuranyl, benzimidazolyl,benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl,benzooxadiazolyl, benzothiadiazolyl, benzotriazolyl, purinyl,quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl.

The term “heterocycloalkyl” denotes a monovalent saturated or partlyunsaturated mono- or bicyclic ring system of 3 to 9 ring atoms,comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, theremaining ring atoms being carbon. In particular embodiments,heterocycloalkyl is a monovalent saturated monocyclic ring system of 4to 7 ring atoms, comprising 1, 2, or 3 ring heteroatoms selected from N,O and S, the remaining ring atoms being carbon. Examples for monocyclicsaturated heterocycloalkyl are aziridinyl, oxiranyl, azetidinyl,oxetanyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydro-thienyl,pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl,thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl,piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl,azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Examples forbicyclic saturated heterocycloalkyl are 8-aza-bicyclo[3.2.1]octyl,quinuclidinyl, 8-oxa-3-aza-bicyclo[3.2.1]octyl,9-aza-bicyclo[3.3.1]nonyl, 3-oxa-9-aza-bicyclo[3.3.1]nonyl, or3-thia-9-aza-bicyclo[3.3.1]nonyl. Examples for partly unsaturatedheterocycloalkyl are dihydrofuryl, imidazolinyl, dihydro-oxazolyl,tetrahydro-pyridinyl, or dihydropyranyl.

The term “hydroxyalkyl” denotes an alkyl group wherein at least one ofthe hydrogen atoms of the alkyl group has been replaced by a hydroxygroup. Examples of hydroxyalky include hydroxymethyl, 2-hydroxyethyl,2-hydroxypropyl, 3-hydroxypropyl, 1-(hydroxymethyl)-2-methylpropyl,2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 2,3-dihydroxypropyl,2-hydroxy-1-hydroxymethylethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutylor 2-(hydroxymethyl)-3 hydroxypropyl.

The term “optional” or “optionally” denotes that a subsequentlydescribed event or circumstance may but need not occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not.

The term “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

Pharmaceutical Compositions and Administration

Another embodiment provides pharmaceutical compositions or medicamentscontaining the compounds of the invention and a therapeutically inertcarrier, diluent or excipient, as well as methods of using the compoundsof the invention to prepare such compositions and medicaments. In oneexample, compounds of formula I may be formulated by mixing at ambienttemperature at the appropriate pH, and at the desired degree of purity,with physiologically acceptable carriers, i.e., carriers that arenon-toxic to recipients at the dosages and concentrations employed intoa galenical administration form. The pH of the formulation dependsmainly on the particular use and the concentration of compound, butpreferably ranges anywhere from about 3 to about 8. In one example, acompound of formula I is formulated in an acetate buffer, at pH 5. Inanother embodiment, the compounds of formula I are sterile. The compoundmay be stored, for example, as a solid or amorphous composition, as alyophilized formulation or as an aqueous solution.

Compositions are formulated, dosed, and administered in a fashionconsistent with good medical practice. Factors for consideration in thiscontext include the particular disorder being treated, the particularmammal being treated, the clinical condition of the individual patient,the cause of the disorder, the site of delivery of the agent, the methodof administration, the scheduling of administration, and other factorsknown to medical practitioners. The “effective amount” of the compoundto be administered will be governed by such considerations, and is theminimum amount necessary to modify FoxM1 gene splicing. For example,such amount may be below the amount that is toxic to normal cells, orthe mammal as a whole.

The compounds of the invention may be administered by any suitablemeans, including oral, topical (including buccal and sublingual),rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal,intrapulmonary, intradermal, intrathecal and epidural and intranasal,and, if desired for local treatment, intralesional administration.Parenteral infusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration.

The compounds of the present invention may be administered in anyconvenient administrative form, e.g., tablets, powders, capsules,solutions, dispersions, suspensions, syrups, sprays, suppositories,gels, emulsions, patches, etc. Such compositions may contain componentsconventional in pharmaceutical preparations, e.g., diluents, carriers,pH modifiers, sweeteners, bulking agents, and further active agents.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier or excipient. Suitable carriers and excipientsare well known to those skilled in the art and are described in detailin, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Formsand Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins,2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice ofPharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe,Raymond C. Handbook of Pharmaceutical Excipients. Chicago,Pharmaceutical Press, 2005. The formulations may also include one ormore buffers, stabilizing agents, surfactants, wetting agents,lubricating agents, emulsifiers, suspending agents, preservatives,antioxidants, opaquing agents, glidants, processing aids, colorants,sweeteners, perfuming agents, flavoring agents, diluents and other knownadditives to provide an elegant presentation of the drug (i.e., acompound of the present invention or pharmaceutical composition thereof)or aid in the manufacturing of the pharmaceutical product (i.e.,medicament).

An example of a suitable oral dosage form is a tablet containing about25 mg, 50 mg, 100 mg, 250 mg, or 500 mg of the compound of the inventioncompounded with about 90-30 mg anhydrous lactose, about 5-40 mg sodiumcroscarmellose, about 5-30 mg polyvinylpyrrolidone (PVP) K30, and about1-10 mg magnesium stearate. The powdered ingredients are first mixedtogether and then mixed with a solution of the PVP. The resultingcomposition can be dried, granulated, mixed with the magnesium stearateand compressed to tablet form using conventional equipment. An exampleof an aerosol formulation can be prepared by dissolving the compound,for example 5-400 mg, of the invention in a suitable buffer solution,e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodiumchloride, if desired. The solution may be filtered, e.g., using a 0.2micron filter, to remove impurities and contaminants.

An embodiment, therefore, includes a pharmaceutical compositioncomprising a compound of Formula I or a stereoisomer or pharmaceuticallyacceptable salt thereof. In a further embodiment includes apharmaceutical composition comprising a compound of Formula I, or astereoisomer or pharmaceutically acceptable salt thereof, together witha pharmaceutically acceptable carrier or excipient.

Another embodiment includes a pharmaceutical composition comprising acompound of formula I for use in the treatment of a hyperproliferativedisease. Another embodiment includes a pharmaceutical compositioncomprising a compound of Formula I for use in the treatment of cancer.

In specific embodiments, the cancer treated by the compounds of thepresent invention is leukemia, acute myeloid leukemia, colon cancer,gastric cancer, macular degeneration, acute monocytic leukemia, breastcancer, hepatocellular carcinoma, cone-rod dystrophy, alveolar soft partsarcoma, myeloma, skin melanoma, prostatitis, pancreatitis, pancreaticcancer, retinitis, adenocarcinoma, adenoiditis, adenoid cysticcarcinoma, cataract, retinal degeneration, gastrointestinal stromaltumor, Wegener's granulomatosis, sarcoma, myopathy, prostateadenocarcinoma, Hodgkin's lymphoma, ovarian cancer, non-Hodgkin'slymphoma, multiple myeloma, chronic myeloid leukemia, acutelymphoblastic leukemia, renal cell carcinoma, transitional cellcarcinoma, colorectal cancer, chronic lymphocytic leukemia, anaplasticlarge cell lymphoma, kidney cancer, breast cancer, cervical cancer.

In specific embodiments, the cancer prevented and/or treated inaccordance with the present invention is basal cell carcinoma, gobletcell metaplasia, or a malignant glioma, cancer of the liver, breast,lung, prostate, cervix, uterus, colon, pancreas, kidney, stomach,bladder, ovary, or brain.

In specific embodiments, the cancer prevented and/or treated inaccordance with the present invention include, but are not limited to,cancer of the head, neck, eye, mouth, throat, esophagus, esophagus,chest, bone, lung, kidney, colon, rectum or other gastrointestinal tractorgans, stomach, spleen, skeletal muscle, subcutaneous tissue, prostate,breast, ovaries, testicles or other reproductive organs, skin, thyroid,blood, lymph nodes, kidney, liver, pancreas, and brain or centralnervous system.

Specific examples of cancers that can be prevented and/or treated inaccordance with present invention include, but are not limited to, thefollowing: renal cancer, kidney cancer, glioblastoma multiforme,metastatic breast cancer; breast carcinoma; breast sarcoma;neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma;malignant melanoma; carcinomas of the epidermis; leukemias such as butnot limited to, acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemias such as myeloblastic, promyelocytic,myelomonocytic, monocytic, erythroleukemia leukemias and myclodysplasticsyndrome, chronic leukemias such as but not limited to, chronicmyelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairycell leukemia; polycythemia vera; lymphomas such as but not limited toHodgkin's disease, non-Hodgkin's disease; multiple myelomas such as butnot limited to smoldering multiple myeloma, nonsecretory myeloma,osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma andextramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonalgammopathy of undetermined significance; benign monoclonal gammopathy;heavy chain disease; bone cancer and connective tissue sarcomas such asbut not limited to bone sarcoma, myeloma bone disease, multiple myeloma,cholesteatoma-induced bone osteosarcoma, Paget's disease of bone,osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant celltumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissuesarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi'ssarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma,rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but notlimited to, glioma, astrocytoma, brain stem glioma, ependymoma,oligodendroglioma, nonglial tumor, acoustic neurinoma,craniopharyngioma, medulloblastoma, meningioma, pineocytoma,pineoblastoma, and primary brain lymphoma; breast cancer including butnot limited to adenocarcinoma, lobular (small cell) carcinoma,intraductal carcinoma, medullary breast cancer, mucinous breast cancer,tubular breast cancer, papillary breast cancer, Paget's disease(including juvenile Paget's disease) and inflammatory breast cancer;adrenal cancer such as but not limited to pheochromocytom andadrenocortical carcinoma; thyroid cancer such as but not limited topapillary or follicular thyroid cancer, medullary thyroid cancer andanaplastic thyroid cancer; pancreatic cancer such as but not limited to,insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secretingtumor, and carcinoid or islet cell tumor; pituitary cancers such as butlimited to Cushing's disease, prolactin-secreting tumor, acromegaly, anddiabetes insipius; eye cancers such as but not limited to ocularmelanoma such as iris melanoma, choroidal melanoma, and cilliary bodymelanoma, and retinoblastoma; vaginal cancers such as squamous cellcarcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamouscell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma,and Paget's disease; cervical cancers such as but not limited to,squamous cell carcinoma, and adenocarcinoma; uterine cancers such as butnot limited to endometrial carcinoma and uterine sarcoma; ovariancancers such as but not limited to, ovarian epithelial carcinoma,borderline tumor, germ cell tumor, and stromal tumor; cervicalcarcinoma; esophageal cancers such as but not limited to, squamouscancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoidcarcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma,verrucous carcinoma, and oat cell (small cell) carcinoma; stomachcancers such as but not limited to, adenocarcinoma, fungating(polypoid), ulcerating, superficial spreading, diffusely spreading,malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; coloncancers; KRAS mutated colorectal cancer; colon carcinoma; rectalcancers; liver cancers such as but not limited to hepatocellularcarcinoma and hepatoblastoma, gallbladder cancers such asadenocarcinoma; cholangiocarcinomas such as but not limited topappillary, nodular, and diffuse; lung cancers such as KRAS-mutatednon-small cell lung cancer, non-small cell lung cancer, squamous cellcarcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinomaand small-cell lung cancer; lung carcinoma; testicular cancers such asbut not limited to germinal tumor, seminoma, anaplastic, classic(typical), spermatocytic, nonseminoma, embryonal carcinoma, teratomacarcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such asbut not limited to, androgen-independent prostate cancer,androgen-dependent prostate cancer, adenocarcinoma, leiomyosarcoma, andrhabdomyosarcoma; penal cancers; oral cancers such as but not limited tosquamous cell carcinoma; basal cancers; salivary gland cancers such asbut not limited to adenocarcinoma, mucoepidermoid carcinoma, andadenoidcystic carcinoma; pharynx cancers such as but not limited tosquamous cell cancer, and verrucous; skin cancers such as but notlimited to, basal cell carcinoma, squamous cell carcinoma and melanoma,superficial spreading melanoma, nodular melanoma, lentigo malignantmelanoma, acrallentiginous melanoma; kidney cancers such as but notlimited to renal cell cancer, adenocarcinoma, hypernephroma,fibrosarcoma, transitional cell cancer (renal pelvis and/or uterer);renal carcinoma; Wilms' tumor; bladder cancers such as but not limitedto transitional cell carcinoma, squamous cell cancer, adenocarcinoma,carcinosarcoma. In addition, cancers include myxosarcoma, osteogenicsarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma,synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,bronchogenic carcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma and papillary adenocarcinomas.

In certain embodiments cancers that can be prevented and/or treated inaccordance with the present invention include, the following: pediatricsolid tumor, Ewing's sarcoma, Wilms tumor, neuroblastoma, neurofibroma,carcinoma of the epidermis, malignant melanoma, cervical carcinoma,colon carcinoma, lung carcinoma, renal carcinoma, breast carcinoma,breast sarcoma, metastatic breast cancer, HIV-related Kaposi's sarcoma,prostate cancer, androgen-independent prostate cancer,androgen-dependent prostate cancer, neurofibromatosis, lung cancer,non-small cell lung cancer, KRAS-mutated non-small cell lung cancer,malignant melanoma, melanoma, colon cancer, KRAS-mutated colorectalcancer, glioblastoma multiforme, renal cancer, kidney cancer, bladdercancer, ovarian cancer, hepatocellular carcinoma, thyroid carcinoma,rhabdomyosarcoma, acute myeloid leukemia, and multiple myeloma.

In certain embodiments, cancers and conditions associated therewith thatare prevented and/or treated in accordance with the present inventionare breast carcinomas, lung carcinomas, gastric carcinomas, esophagealcarcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas,thecomas, arrhenoblastomas, cervical carcinomas, endometrial carcinoma,endometrial hyperplasia, endometriosis, fibrosarcomas, choriocarcinoma,head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas,hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma,cavernous hemangioma, hemangioblastoma, pancreas carcinomas,retinoblastoma, astrocytoma, glioblastoma, Schwannoma,oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, renal cell carcinoma, prostate carcinoma,abnormal vascular proliferation associated with phakomatoses, edema(such as that associated with brain tumors), or Meigs' syndrome. Inspecific embodiment, the cancer an astrocytoma, an oligodendroglioma, amixture of oligodendroglioma and an astrocytoma elements, an ependymoma,a meningioma, a pituitary adenoma, a primitive neuroectodermal tumor, amedullblastoma, a primary central nervous system (CNS) lymphoma, or aCNS germ cell tumor.

In specific embodiments, the cancer treated in accordance with thepresent invention is an acoustic neuroma, an anaplastic astrocytoma, aglioblastoma multiforme, or a meningioma.

In other specific embodiments, the cancer treated in accordance with thepresent invention is a brain stem glioma, a craniopharyngioma, anependyoma, a juvenile pilocytic astrocytoma, a medulloblastoma, an opticnerve glioma, primitive neuroectodermal tumor, or a rhabdoid tumor.

Preparation of Compound 3

Step A: To a solution of diethyl 1H-pyrazole-3,5-dicarboxylate (10.0 g,47 mmol) and chloroacetone (3.76 mL, 47 mmol) in acetone (200 mL) wasadded potassium carbonate (7.2 g, 52 mmol). After heating at 30° C. for6 h, the mixture was concentrated to remove the volatiles. The residuewas taken into EtOAc and washed with water. The organics were dried overMgSO4 and concentrated to give diethyl1-(2-oxopropyl)-1H-pyrazole-3,5-dicarboxylate as a light brown solid,which was used directly in the next step, MS m/z 269.1 [M+H]+.

Step B: To a solution of diethyl1-(2-oxopropyl)-1H-pyrazole-3,5-dicarboxylate (˜47 mmol) in acetic acid(300 mL) was added ammonium acetate (72 g, 940 mmol). After refluxingfor 48 h, the mixture was concentrated to minimum volume and dilutedwith water. The precipitate was filtered, washed with water and MeCN togive ethyl 4-hydroxy-6-methylpyrazolo[1,5-a]pyrazine-2-carboxylate as atan solid (6.7 g, 64%), MS m/z 222.1 [M+H]+.

Step C: A mixture of ethyl4-hydroxy-6-methylpyrazolo[1,5-a]pyrazine-2-carboxylate (7.18 g, 32.5mmol) in POCl3 (80 mL) was refluxed for 15 h. The dark mixture wasconcentrated and washed with MeCN to give ethyl4-chloro-6-methylpyrazolo[1,5-a]pyrazine-2-carboxylate (5.197 g) as anoff-white solid. The filtrate was concentrated and chromatographed togive an additional 1.42 g product (6.617 g, 85%) MS m/z 240.1 [M+H]+,242.1 [M+2+H]+.

Step D: A mixture of ethyl4-chloro-6-methylpyrazolo[1,5-a]pyrazine-2-carboxylate (5.197 g, 21.7mmol), MeB(OH)2 (3.90 g, 65.1 mmol), K2CO3 (14.8 g, 107.5 mmol) andPd(PPh3)2Cl2 in (456 mg, 0.65 mmol) DMF (100 mL) was degassed and heatedunder N2 for 15 h. The mixture was concentrated on a rotovap to removemost of the DMF and washed with water. The residue was chromatographed(2% to 5% MeOH in CH2Cl2) to give ethyl4,6-dimethylpyrazolo[1,5-a]pyrazine-2-carboxylate as a yellow solid(3.90 g, 82%), MS m/z 220.1 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ 8.54(1H, s), 7.49 (1H, s), 4.36 (2H, q, J=7.2 Hz), 2.70 (3H, s), 2.42 (3H,s), 1.34 (3H, t, J=7.2 Hz).

Step E: To a solution of t-butyl acetate (1.63 mL, 12.1 mmol) in THF (50mL) at −78° C. was added LDA (1.5 M, 0.97 mL, 14.5 mmol). After 0.5 h,the solution was cannulated to a solution of ethyl4,6-dimethylpyrazolo[1,5-a]pyrazine-2-carboxylate (1.33 g, 6.07 mmol) inTHF (100 mL) at −30° C. After 1 h, the mixture was quenched withsaturated NH4Cl, adjusted to pH 5-6 and extracted with EtOAc. Thecombined organics were dried and concentrated. The residue waschromatographed (2% to 4% MeOH/CH2Cl2) to give t-butyl3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-3-oxopropanoate as a yellowoil (1.696 g, 97%), MS m/z 290.2 [M+H]+; 1H NMR (500 MHz, DMSO-d6): δ8.57 (1H, s), 7.50 (1H, s), 4.02 (2H, s), 2.70 (3H, s), 2.43 (3H, s),1.38 (9H, s).

Step F: A solution of t-butyl3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-3-oxopropanoate (4.86 g,16.8 mmol) in EtOH (30 mmol) was heated at 120° C. in a capped tube.After 1 h, the solution was cooled to rt and the volatiles were removedto give ethyl3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-3-oxopropanoate as a yellowsolid (4.44 g, 98%), MS m/z 262.2 [M+H]+.

Step G: A mixture of 2-amino-5-fluoro-pyridine (134 mg, 1.2 mmol), ethyl3-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-3-oxopropanoate (261 mg, 1.0mmol) and PPTs (12.6 mg, 0.05 mmol) was heated at 130° C. After 8 h, themixture was cooled to rt and chromatographed to give2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-oneas a yellow solid (220 mg, 71%). MS m/z 310.2 [M+H]+; 1H NMR (500 MHz,DMSO-d6): δ 8.97-8.95 (1H, m), 8.55 (1H, s), 8.16-8.12 (1H, m),7.87-7.85 (1H, m), 7.56 (1H, s), 7.03 (1H, s), 2.73, (3H, s), 2.43 (3H,s).

Step H:2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-fluoro-4H-pyrido[1,2-a]pyrimidin-4-one(309 mg, 1.0 mmol) and piperazine (1.1 mL, 10 mmol) in DMA (1.0 mL) washeated at 120° C. After 15 h, the volatiles were removed and the residuewas washed with MeCN to give the title compound as a yellow solid (313mg, 80%). M.P. 254-256° C.; MS m/z 390.4 [M+H]+; 1H NMR (500 MHz,DMSO-d6): δ 8.55 (1H, s), 8.27 (1H, d, J=2.7 Hz), 8.12 (1H, dd, J=2.8Hz, 9.7 Hz), 7.71 (1H, d, J=9.7 Hz), 7.54 (1H, s), 6.95 (1H, s), 3.25(4H, m), 2.72 (3H, s), 2.51 (4H, m, obscured by DMSO-d6), 2.43 (3H, s),2.25 (3H, s).

Additional compounds disclosed herein may be prepared according to theabove example by substituting the appropriate starting materials,reagents and reaction conditions.

WO 2013/119916 discloses additional compounds which can be used forprophylaxis or treatment of cancer by FoxM1 splicing modification. WO2013/119916 is hereby included by reference.

The term “M.P.” represents “Melting Point (° C.),” the term “MS”represents “Mass Spectroscopy Peak(s) m/z [M+H]⁺, [M+2+H]⁺, [M−H]⁻ or[M+2−H]⁻,” the term “D” represents “Decomposition/Decomposed”.

Com- pound Compound Name M.P. MS 12-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)- 231-240 417.47-(1-ethylpiperidin-4-yl)-9-methyl-4H- pyrido[1,2-a]pyrimidin-4-one 22-(4-ethyl-6-methylpyrazolo[1,5-a]pyrazin- 265 (D) 401.32-yl)-9-methyl-7-(1,2,3,6-tetrahydropyridin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one 32-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2- 254-256 390.4yl)-7-(4-methylpiperazin-1-yl)-4H- pyrido[1,2-a]pyrimidin-4-one 42-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2- 195-200 376.5yl)-7-(piperazin-1-yl)-4H-pyrido[1,2- a]pyrimidin-4-one

EXAMPLES Example 1: Cpds. Induce Alternative Splicing of FoxM1 TowardsFoxM1A

To investigate an effect on splicing of FoxM1, human fibroblasts weretreated for 24 hours with cpds. 1-4 in dose response, and analysed byRT-qPCR for presence of mRNA including (FoxM1A) or excluding (FoxM1B/C)the Δ9 exon. The resulting dose response curves were fitted to a Hillbinding equation. FIG. 1A shows that all cps. increased expression ofthe FoxM1A mRNA including exon 9, and EC₅₀ values were calculated to0.246, 0.016, 1.210 and 0.068 uM for cpds. 1, 2, 3 and 4, respectively.Correspondingly, the mRNAs for the FoxM1B/C isoforms lacking exon 9 (Δ9versions) declined with EC₅₀ values of 0.724, 0.014, 3.541 and 0.104 uMfor cpds. 1, 2, 3 and 4, respectively. Correlation analysis of the EC₅₀values for upregulation of FoxM1A and downregulation of FoxM1B/Crevealed an excellent linear correlation (r²=0.992) with no obviousshift from the line of identity (FIG. 1C). The data suggest a close anddirect functional relation of upregulation of FoxM1A and downregulationof FoxM1B/C.

Example 2: Alternative Splicing Towards FoxM1A Induces Cytotoxicity

For investigation of the effect of altered FoxM1 expression onmicronucleus induction (MNT) and and cell survival, human fibroblastswere treated for up to 120 hours with cpds. 1-4 in dose response, andMNT, cytotoxicity or Cell Index were assessed on the xCELLigenceplatform, monitoring proliferation rates and cell death online. Todefine a quantitative measure for cytotoxic effects, a 2% cut-off forMNT induction (FIG. 2A), a 75% cut-off for cell survival (EC_(75%)), anda 75% cut-off was estimated (ED_(75%)). Cpd. 2 and 4 were most potent inMNT, cytotoxicity and reduction of Cell Index (FIGS. 2A, 2B and 2C,respectively). The data suggest that induction of alternative splicingof FoxM1 towards FoxM1A induced slowing of proliferation and cell death.

Example 3: Alternative Splicing of FoxM1 Correlates with Cytotoxicity

Correlation of the 2%, EC_(75%) and ED_(75%) values for the induction inMNT, cytotoxicity or reduction in Cell Index to the EC₅₀ for FoxM1B/Creduction (FIGS. 3A, 3B and 3C, respectively) revealed excellent linearcorrelations, best for the Cell Index (r²=0.963). Thereby, a ˜10-foldshift in activity indicated that concentrations of 10-fold above theEC₅₀ for FoxM1B/C reduction were required to reach the ED_(75%) (FIG.3C).

Example 4: Alternative Splicing of FoxM1 Correlates with Cytotoxicity

Correlation of the 2%, EC_(75%) and ED_(75%) values for the induction inMNT, cytotoxicity or reduction in Cell Index to the EC₅₀ for FoxM1B/Creduction (FIGS. 4A, 4B and 4C, respectively) revealed excellent linearcorrelations, best for the Cell Index (r²=0.951), and a 10- to 15-foldshift in the activity indicated that concentrations of 10- to 15-foldabove the EC₅₀ for FoxM1A induction were required to reach the ED_(75%)(FIG. 4C).

The data of examples 3 and 4 suggest that a >90 shift in FoxM1 splicingfrom FoxM1B/C towards FoxM1A is required to induce meaningful reductionin Cell Index as measure for cell proliferation and survival.

Example 5: Increase of FoxM1A Protein Correlates with Cytotoxicity

To assess if alternative splicing of FoxM1 towards FoxM1A results inmeaningful changes in the protein levels and cell death, human primarymyoblasts were treated under proliferating conditions to modulate FoxM1expression and evaluate its consequence. Under proliferative conditions,FoxM1A was detectable by Western Blot, but at low concentrations (FIG.4A). Treatment with cpd. 3 at doses up to 10 μM strongly reduced proteinlevels of actin, a direct marker of cell numbers, but increased proteinlevels of FoxM1A (FIG. 5A). Quantitative analysis of protein levelsindicated that actin was reduced by 6-fold compared to controls (FIG.5B). When normalized to actin, FoxM1A protein levels increased by30-fold at the highest dose (FIG. 5C). The data suggest that inproliferating myoblasts, alternative splicing towards FoxM1A increasedFoxM1A protein and induced cell death.

Example 6: No Change of FoxM1A Protein in Non-Proliferative Conditions

To assess if alternative splicing of FoxM1 towards FoxM1A is presentalso in cells that are not proliferating, the same human primarymyoblasts were differentiated to investigate if FoxM1 expression is alsopresent and can be modulated similarly under those conditions. Underdifferentiating conditions, FoxM1A was undetectable by Western Blot(FIG. 6A). Treatment with cpd. 3 at doses up to 10 μM did not show anyreduction in protein levels of actin as marker of cell numbers (FIG.6A). Quantitative analysis of protein levels indicated that actin wasnot altered compared to controls (FIG. 6B), whereas FoxM1A levelsnormalized to actin was undetectable. The data suggest that FoxM1Aexpression is restricted to proliferating cells, and alternativesplicing towards FoxM1A does not induce cell death in cells that are notproliferating.

Example 7: Increase of FoxM1A Protein Induces Cytotoxicity in BreastCancer Cells

Upregulation of FoxM1A protein correlates with cytotoxicity in breastcancer cells. Human BT474 breast cancer cells were treated with cpd. 2at 10 μM for 1 or 2 days under proliferating conditions (in the presenceof serum), and total protein extracts analyzed by SDS PAGE and WesternBlot for FoxM1A and actin protein levels. To assess if increase inFoxM1A protein by alternative splicing of FoxM1 towards FoxM1A inducescell death in a cancer condition, human breast cancer cells (BT474) weretreated to modulate FoxM1 expression, and FoxM1A protein levels wereassessed at day 1 and 2 of treatment. On day 1 and 2 under controlconditions, FoxM1A was detectable by Western Blot, but at lowconcentrations. Treatment with cpd. 2 at 10 μM did not have any effecton FoxM1A protein but slightly reduced actin protein on day 1, butstrongly reduced actin protein on day 2, with a concomitant increase inFoxM1A protein levels. Quantitative analysis of protein levels indicatedthat actin was reduced on day 1 by 18%, and by more than 90% on day 2 bytreatment with cpd. 2 at 10 μM, whereas FoxM1A levels were increased inthe same samples by nearly 3-fold. When normalized to actin, FoxM1Aprotein levels increased by 28-fold by treatment with cpd. 2 at 10 μM.The data suggest that in breast cancer cells, alternative splicingtowards FoxM1A increased FoxM1A protein and induced cell death. Datarepresent means±SEM of 3 independent observations. Statisticalcomparison was performed by one-way ANOVA followed by Dunnet's post-hoctest. *, p<0.05, ***, p<0.001.

Methods

Monitoring Expression Levels of FoxM1 Splice Variants Using Real-TimeQuantitative PCR.

Fibroblasts, at 10000 cells per cm² were treated with varying doses ofcompounds (0.01-10 μM) for 24 hours. RNA extraction was performed as perinstructions mentioned in the Ambion® Cells-to-CT™ Kits from AppliedBiosystems®. RNA samples were frozen at −20° C. until further analysis.Relative expression levels of FoxM1A or FoxM1B/C along with GAPDH forinternal control, was measured using one-step multiplex reversetranscription-polymerase chain reaction (RT-PCR). TaqMan® FAM probeswere used for relative quantitation of FoxM1A or FoxM1B/C expressionlevels and TaqMan® VIC probes were used for relative quantitation ofhuman GAPDH levels. The fidelity of the amplification methods wasdetermined using the ΔΔCt relative quantification method forquantitative PCR.

Monitoring Real-Time Effects on Fibroblast Cell Proliferation as Well asToxicity

Fibroblasts at 10000 cells per cm² were treated with varying doses ofcompounds (0.1-10 μM) for 5 days in an xCELLigence E Plate-16 format.Plates were transferred onto the xCELLigence RTCA-DP instrument placedin the 37° C., 5% CO₂ incubator and background impedance measurement ofall the wells was recorded. Fibroblasts were seeded into the wells andincubated for approximately 5 hours to facilitate even spreading andstabilization of the cells. Changes in impedance at the goldmicroelectrodes covering the under-surface of the membranes as the cellsattach and spread was measured and recorded every 30 minutes over 120hours (5 days). Impedance was represented by the relative anddimensionless parameter named Cell Index (CI). Cell Indexvalues=Zt−Zi/15 [Ohm]; where Zi=initial impedance at the start of theexperiment and Zt=individual time-points during the experiment (A. K.Bosserhoff, L. Ellmann, S. Kuphal. S: Melanoblasts in culture as an invitro system to determine molecular changes in melanoma. 2011.Experimental Dermatology, 20, 435-440). The values obtained in theinitial six hours were annulled from the slope-calculations to take intoconsiderations any variations observed due to the differences, if any,in the attachment capabilities of cells in response to the treatments.

Human Myoblast or Breast Cancer Cell Culture and Western Blot Analysis

Human myoblasts were acquired from ECACC, BT474 cells were obtained fromATCC, and were cultivated according to supplier protocols. Forexperimental purpose, human myoblasts were cultivated for 5 days andwere treated with varying doses of compounds (0.1-10 μM). BT474 cellswere cultivated for up to 2 days and treated with compounds at 10 μM.For Western blot analyses, myoblast cells treated over 5 days or BT474cells treated for 2 days were lysed in boiling Laemmli buffer (Bio-Rad)containing 100 mM dithiothreitol. SDS PAGE blots were probed withrabbit-anti FoxM1 antibody (Cell Signaling Technology, 1:1000), goatanti-Actin (Santa Cruz Biotechnology, 1:20000) and Alexa680/800secondary antibodies (Molecular Probes, 1:10,000). Fluorescence wasacquired with the Odyssey imaging system (Licor Biosciences), FoxM1Aintensity was normalized for actin. Data were analyzed using GraphPadsoftware.

The invention claimed is:
 1. A method for the treatment of cancer in asubject in need thereof comprising administering to the subject aneffective amount of a compound selected from the group consisting of:2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(1-ethylpiperidin-4-yl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one;2-(4-ethyl-6-methylpyrazolo[1,5-a]pyrazin-2-yl)-9-methyl-7-(1,2,3,6-tetrahydropyridin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one;2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one;and2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(piperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one,or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the cancer is a cancer expressing a FoxM1 gene.
 3. The method ofclaim 1, wherein the subject is a human.
 4. The method of claim 3,wherein the cancer is a cancer expressing a human FoxM1 gene.
 5. Themethod of claim 1, wherein the cancer is selected from the groupconsisting of cancers of the liver, prostate, brain, breast, lung,colon, pancreas, skin, cervix, ovary, mouth, blood and nervous system.6. The method of claim 1, wherein the compound is2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(1-ethylpiperidin-4-yl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one,or a pharmaceutically acceptable salt thereof.
 7. The method of claim 1,wherein the compound is2-(4-ethyl-6-methylpyrazolo[1,5-a]pyrazin-2-yl)-9-methyl-7-(1,2,3,6-tetrahydropyridin-4-yl)-4H-pyrido[1,2-a]pyrimidin-4-one,or a pharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the compound is2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(4-methylpiperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one,or a pharmaceutically acceptable salt thereof.
 9. The method of claim 1,wherein the compound is2-(4,6-dimethylpyrazolo[1,5-a]pyrazin-2-yl)-7-(piperazin-1-yl)-4H-pyrido[1,2-a]pyrimidin-4-one,or a pharmaceutically acceptable salt thereof.